PROJECT SUMMARY Pneumococcal conjugate vaccines (PCVs), composed of a pneumococcal polysaccharide covalently linked to a carrier protein, are life-saving prophylactics used to prevent pneumococcal disease. Importantly, PCVs provide immunity for all age groups, including, infants and children under the age of two, which is not the case for purely polysaccharide vaccines. Like all conjugate vaccines, PCVs are manufactured using chemical conjugation, which is notoriously complex, labor intensive, and ultimately hinders the development of better versions that provide immunity to more disease-causing serotypes. As an example, the PCV, Prevnar 13, was licensed in 2010 and only protects against 13 pneumococcal serotypes; whereas, the purely polysaccharide vaccine, Pneumovax 23, was licensed in 1983 and protects against 23 serotypes. Pneumovax 23 is approved for use in the elderly; however, it does not provide protection to infants and children. Thus, for more than three decades, infants and children have not had a vaccine option that protects against 20+ disease causing pneumococcal serotypes. In order to provide a 20+ valent PCV for use in all age groups, VaxNewMo has been developing a method for manufacturing pneumococcal and other conjugate vaccines that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems in process termed bioconjugation. VaxNewMo?s proprietary bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide, like a pneumococcal capsular polysaccharide, to a carrier protein all within the lab safe bacterium E. coli. Since bioconjugation is an enzyme driven technology, the conjugates produced are homogenous and readily purified. Importantly, bioconjugation can be used to rapidly produce a plethora of conjugates against many serotypes simply by introducing the genetic information encoding for a different pneumococcal serotype into a bioconjugation competent strain of E. coli. Thus, bioconjugation can be used for the streamlined development of a PCV covering more than 20 serotypes. In Phase I of this project, we successfully established proof of principle that VaxNewMo?s bioconjugation platform could generate PCVs containing conventional vaccine carriers. Moreover, VaxNewMo?s PCVs were both immunogenic and protective against pneumococcal disease. The proposed research in this Phase II SBIR application will focus on (Aim 1) establishing bioprocessing capabilities for large volumetric production of VNM8, a serotype 8 pneumococcal bioconjugate. Establishing bioprocessing procedures for a single serotype bioconjugate is an important first step towards commercial scale production and will help streamline future upstream processing for other pneumococcal bioconjugates. Subsequently, (Aim 2) we will confirm that VNM8 produced in a large volumetric bioprocess is protective using in vitro and in vivo models. In addition, (Aim 3) we will generate a library of E. coli strains capable of producing a 24 valent PCV. For Phase IIB, will seek to formulate a 24 valent PCV (24vPneumo) for pharmacokinetic and toxicity studies and prepare for pre-IND meetings with the FDA.